Chemical Modification of the Mitochodrial bc₁ by N,N′ ‐Dicyclohexylcarbodiimide Inhibits Proton Translocation

Abstract

We report here that N,N/i′‐dicyclohexylcarbodiimide (DCCD) decreases the H2e stoichiometry of the mytochrome bc1 complex from 308 ± 0.2 (10) to 201 ± 0.1 (8) but has only a minimal effect on the H2e ratio of cytochrome oxidase under the relatively mild condition used. The veffect on the bc1 complex connot be explained by uncoupling, by inhibition of electron transport or by selective mitochondrial damage. We conclude that DCCD is an inhibitor of proton translocation within ther bc1 complex. There are three possible explanation of this effect: (a) DCCD could alter the pathway of electron flow, (b) DCCD could prevent one of the proton translocation reations but not electrton transport, (c) DCCD could prevent the couduction of the translocated proton to the external phase.

The denaturation of rabbit muscle phosphorylase b by guanidinium chloride

“…Phosphorylase b is clearly much more sensitive to denaturation by GdnHCl than are a number of other oligomeric enzymes, such as lactate dehydrogenase (Fig. 1), creatine kinase (Price & Stevens, 1982), pyruvate kinase (Price & Stevens, 1983) and phosphoglycerate mutase (N. C. Price, unpublished work). The substantial inactivation brought about by 0.2M-GdnHCl (Fig.…”

“…LDH-5 isoenzyme (Fig. 1), rabbit muscle creatine kinase (Bickerstaff et al, 1980), rabbit muscle pyruvate kinase (Price & Stevens, 1983) and yeast phosphoglycerate mutase (N. C. Price, unpublished work) none of which is inactivated to any measurable extent by prolonged incubation in 0.1 M-GdnHCl. As also shown in Fig.…”

“…A commonly used procedure to study the refolding of an enzyme is to dilute a solution of denatured enzyme to such an extent that the residual concentration of denaturing agent is without effect on the properties of native enzyme (see, e.g., Bickerstaff et al, 1980;Jaenicke et al, 1981;Price & Stevens, 1983). The results in Fig.…”

“…It was also noted that irreversible denaturation of the protein could be brought about by agents such as urea or GdnHCl. As part of a series of experiments (Price & Stevens, 1982, 1983 Abbreviations used: GdnHCl, guanidinium chloride;…”

Thermodynamic and steady-state-kinetic investigation of the effect of NN′-dicyclohexylcarbodi-imide on H+ translocation by the mitochondrial cytochrome bc1 complex

“…(3) DCCD acts by increasing proton permeability, to which the thermodynamic analysis is relatively insensitive. It is clear that, at the concentrations we have used, DCCD does act in this way to a small extent (see Table 2 and Price & Brand, 1983). However, we have shown that addition of sufficient FCCP to give the same increase in J0 as given by DCCD has no effect on the measured H+/2e ratio in either oxygen-pulse (Price & Brand, 1983) or steady-state kinetic determinations (Al- Shawi & Brand, 1981).…”